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. 2006 Apr;80(7):3395-405.
doi: 10.1128/JVI.80.7.3395-3405.2006.

Analyses of subgenomic promoters of Hibiscus chlorotic ringspot virus and demonstration of 5' untranslated region and 3'-terminal sequences functioning as subgenomic promoters

Weimin Li  1 , Sek-Man Wong
Affiliations

Analyses of subgenomic promoters of Hibiscus chlorotic ringspot virus and demonstration of 5' untranslated region and 3'-terminal sequences functioning as subgenomic promoters

Weimin Li et al. J Virol. 2006 Apr.

Abstract

Hibiscus chlorotic ringspot virus (HCRSV), which belongs to the genus Carmovirus, generates two 3'-coterminal subgenomic RNAs (sgRNAs) of 1.4 kb and 1.7 kb. Transcription start sites of the two sgRNAs were identified at nucleotides (nt) 2178 and 2438, respectively. The full promoter of sgRNA1, a 118-base sequence, is localized between positions +6 and -112 relative to its transcription start site (+1). Similarly, a 132-base sequence, from +6 to -126, defines the sgRNA2 promoter. Computer analysis revealed that both sgRNA promoters share a similar two-stem-loop (SL1 + SL2) structure, immediately upstream of the transcription start site. Mutational analysis of the primary sequence and secondary structures showed further similarities between the two subgenomic promoters. The basal portion of SL2, encompassing the transcription start site, was essential for transcription activity in each promoter, while SL1 and the upper portion of SL2 played a role in transcription enhancement. Both the 5' untranslated region (UTR) and the last 87 nt at the 3' UTR of HCRSV genomic RNA are likely to be the putative genomic plus-strand and minus-strand promoters, respectively. They function well as individual sgRNA promoters to produce ectopic subgenomic RNAs in vivo but not to the same levels of the actual sgRNA promoters. This suggests that HCRSV sgRNA promoters share common features with the promoters for genomic plus-strand and minus-strand RNA synthesis. To our knowledge, this is the first demonstration that both the 5' UTR and part of the 3' UTR can be duplicated and function as sgRNA promoters within a single viral genome.

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Figures

FIG. 1.
FIG. 1.
Time course of accumulation of HCRSV sgRNA1 and sgRNA2 in kenaf protoplasts. (A) Schematic diagram of the HCRSV genome. The open boxes represent ORFs with encoded protein products. Three single horizontal lines represent gRNA, sgRNA1, and sgRNA2, respectively. Numbers indicate the nucleotide positions on the HCRSV genome. (B) Northern blot analysis of total RNA from kenaf protoplasts transfected with wild-type HCRSV in vitro transcripts. The protoplasts were harvested at the indicated time intervals postinoculation. Bands corresponding to HCRSV gRNA and the sgRNA1 and sgRNA2 are shown with arrows. The level of 28S rRNA was shown to indicate the relative amount of samples loaded.
FIG. 2.
FIG. 2.
Modifications of transcription start sites of HCRSV sgRNA1 and sgRNA2. (A) Mutagenesis of the putative transcription start sites for sgRNA1 and sgRNA2. The nucleotides are shown in the boxes with mutations in boldface and italics, and the mutant names are listed on the left of each box. (B) Northern blot analysis of total RNA from kenaf protoplasts transfected with p223 and mutant transcripts (24 hpi). Bands corresponding to gRNA and sgRNAs are indicated.
FIG. 3.
FIG. 3.
Sequence requirements for ectopic expression of the sgRNA1 promoter. (A) Schematic diagram of constructs in which sequence encompassing the context of sgRNA1 transcription start site was duplicated and inserted into the BglII and SphI sites in the coat protein ORF of the p223BS mutant. Position 2178 is shown as the sgRNA1 transcription start site. Three horizontal lines with original position numbers denote the length of the duplicated sequences, which are indicated with filled arrowheads in the construct. (B) Northern blot analysis of sgRNA accumulation in protoplasts (24 hpi) transfected with in vitro transcripts of p223 and the three test constructs. Bands corresponding to the ectopic sgRNA3 are indicated.
FIG. 4.
FIG. 4.
Mapping of the boundary of sgRNA1 promoter by ectopic expression of sgRNA3. (A) Map of the constructs that contain duplicated copies of the sgRNA1 promoter (filled gray arrow) inserted into the BglII and SphI sites of p223BS. The transcription start site at position 2178 is shown by an arrow. The duplicated sequences are indicated with horizontal solid lines, along which the listed numbers are the original position numbers of 5′ and 3′ ends of the duplicated sequences. The dotted lines represent the deleted sequences. Each deletion mutant is named with the position numbers of two endpoints of the duplicated sequence. (B) Northern blot analysis of the 5′ border of sgRNA1 promoter in kenaf protoplasts. (C) Mapping of the 3′ border of sgRNA1 promoter in kenaf protoplasts. Bands corresponding to gRNA, sgRNA1, sgRNA2, and ectopic sgRNA3 are indicated.
FIG. 5.
FIG. 5.
Mapping of the boundary of sgRNA2 promoter. (A) Schematic representation of deletion mutants. The transcription start site at position 2438 is shown by an arrow. The dotted lines represent the deleted regions. The potential promoter region within each mutant is shown as a horizontal solid line. The numbers denote its 5′ and 3′ positions. (B) Northern blot analysis of the 5′ border of sgRNA2 promoter in kenaf protoplasts. (C) Mapping of the 3′ border of sgRNA2 promoter in kenaf protoplasts. Bands corresponding to gRNA, sgRNA1, and sgRNA2 are indicated.
FIG. 6.
FIG. 6.
Analyses of RNA sequence and secondary structure of sgRNA1 promoter. (A) Mutations in the regions of the duplicated sgRNA1 promoter. The putative two-stem-loop structure (1SL1 and 1SL2) was predicated by MFOLD at 25°C. Boxes contain nucleotides inserted as substitutions into the corresponding boxed structures in the sgRNA1 promoter. The mutant names are listed above the boxes. In 1SL2, helices are designated as 1A, 1B, and 1D; the central region is designated as 1C; and the apical loop is designated as 1E. The right-angled arrow indicates the transcription start site (nt 2178) of sgRNA1. (B and C) Ectopic sgRNA3 syntheses of the mutants were analyzed by Northern blotting. Positions of gRNA, sgRNA1, sgRNA2, and sgRNA3 are indicated.
FIG. 7.
FIG. 7.
Analyses of RNA sequence and secondary structure of sgRNA2 promoter. (A) Mutations in the 2SL1 and 2SL2 regions. The sequence is in the minus strand. In 2SL2, helices are designated as 2A, 2B, and 2D; the central region is designated as 2C; and the apical loop is designated as 2E. The transcription start site (nt 2438) of sgRNA2 is indicated with a right-angled arrow. (B and C) Northern blot analyses of the activity of sgRNA2 promoter mutants. All designations and methods are as described for Fig. 6. Positions of gRNA, sgRNA1, sgRNA2, and sgRNA3 are indicated.
FIG. 8.
FIG. 8.
Sequence and structure-specific requirements of sgRNA1 and sgRNA2 promoters. (A) RNA sequences and secondary structures of the minimal promoters for sgRNA1 and sgRNA2. The right-angled arrow represents the transcription start site (position +1). The numbers, relative to the native transcription start site, indicate the original positions of nucleotides within the promoter regions. The dashed line separates the promoter into two portions, which indicate that the minimal promoter is composed with two distal sequences of the original promoter. (B) Northern blot analysis of the activity of mini-sg1 and mini-sg2 promoter. Positions of gRNA, sgRNA1, sgRNA2, and sgRNA3 are indicated.
FIG. 9.
FIG. 9.
The 5′ UTR and the last 87 nt at the 3′ UTR of HCRSV function as sgRNA promoters. (A) Diagram of constructs in which the sequence of the 5′ UTR (nt 1 to 30) or the last 87 nt of the 3′ UTR (nt 3825 to 3911) was duplicated and inserted into the BglII and SphI sites in the CP ORF of the p223BS mutant. The orientation of the inserted sequence is shown. (B) Northern blot analysis of sgRNA accumulation in protoplasts (24 hpi) transfected with in vitro transcripts of p5′UTR and p3′UTR. Bands corresponding to the ectopic sgRNA3 are indicated.
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